Antibody purification

ABSTRACT

This invention relates to the application of hydrophobic interaction chromatography combination chromatography to the purification of antibody molecule proteins.The questions raised in reexamination request, 09/006,966 filed Mar. 12, 2004 have been considered and the results thereof are reflected in this reissue patent which constitutes the reexamination certificate required by 35 U.S.C. 307 as provided in 37 CFR 1.570( e ) , for ex parte reexaminations, or the reexamination certificate required by 35 U.S.C. 316 as provided in 37 CFR 1.997 ( e )  for inter partes reexaminations.

More than one reissue application has been filed for the reissue of Pat.No. 5,429,746. The reissue applications are application Ser. No.11/140,525 (the present reissue) and 11/804,729 which is a continuationof the reissue application Ser. No. 11/140,525.

FIELD OF THE INVENTION

This invention relates to the field of protein purification. Morespecifically, this invention relates to the application of HydrophobicInteraction Chromatography (HIC) to the separation of Immunoglobulin Gmonomers and to the integration of HIC into a combinationchromatographic protocol for the purification of IgG antibody molecules.

BACKGROUND OF THE INVENTION

Historically, protein purification schemes have been predicated ondifferences in the molecular properties of size, charge and solubilitybetween the protein to be purified and undesired protein contaminants.Protocols based on these parameters include size exclusionchromatography, ion exchange chromatography, differential precipitationand the like.

Size exclusion chromatography, otherwise known as gel filtration or gelpermeation chromatography, relies on the penetration of macromoleculesin a mobile phase into the pores of stationary phase particles.Differential penetration is a function of the hydrodynamic volume of theparticles. Accordingly, under ideal conditions the larger molecules areexcluded from the interior of the particles while the smaller moleculesare accessible to this volume and the order of elution can be predictedby the size of the protein because a linear relationship exists betweenelution volume and the log of the molecular weight.

Chromatographic supports based on cross-linked dextrans e.g. SEPHADEX®,spherical agarose beads e.g. SEPHAROSE® (both commercially availablefrom Pharmacia AB. Uppsala, Sweden), based on cross-linkedpolyacrylamides e.g. BIO-GEL ® (commercially available from BioRadLaboratories, Richmond, Calif.) or based on ethylene glycol-methacrylatecopolymer e.g. TOYOPEARL HW65 (commercially available from Toso HaasCo., Tokyo, Japan) are useful in forming the various chromatographiccolumns for size exclusion, or HIC chromatography in the practice ofcertain aspects of this invention.

Precipitation methods are predicated on the fact that in crude mixturesof proteins the solubilities of individual proteins are likely to varywidely. Although the solubility of a protein in an aqueous mediumdepends on a variety of factors, for purposes of this discussion it canbe said generally that a protein will be soluble if its interaction withthe solvent is stronger than its interaction with protein molecules ofthe same or similar kind. Without wishing to be bound by any particularmechanistic theory describing precipitation phenomena, it is nonethelessbelieved that interaction between a protein and water molecules canoccur by hydrogen bonding with several types of uncharged groups and/orelectrostatically, as dipoles, with charged groups and that precipitantssuch as salts of monovalent cations (e.g. ammonium sulfate) compete withproteins for water molecules. Thus at high salt concentrations, theproteins become “dehydrated” reducing their interaction with the aqueousenvironment and increasing the aggregation with like or similarproteins, resulting in precipitation from the medium.

Ion exchange chromatography involves the interaction of chargedfunctional groups in the sample with ionic functional groups of oppositecharge on an adsorbent surface. Two general types of interaction areknown. Anionic exchange chromatography is mediated by negatively chargedamino acid side chains (e.g., aspartic acid and glutamic acid)interacting with positively charged surfaces and cationic exchangechromatography is mediated by positively charged amino acid residues(e.g., lysine and arginine) interacting with negatively chargedsurfaces.

More recently affinity chromatography and hydrophobic interactionchromatography techniques have been developed to supplement the moretraditional size exclusion and ion exchange-chromatographic protocols.Affinity chromatography relies on the specific interaction of theprotein with an immobilized ligand. The ligand can be specific for theparticular protein of interest in which case the ligand is a substrate,substrate analog, inhibitor, receptor or antibody. Alternatively, theligand may be able to react with a number of related proteins. Suchgroup specific ligands as adenosine monophosphate, adenosinediphosphate, nicotine adenine dinucleotide or certain dyes may beemployed to recover a particular class of proteins.

With respect to the purification of antibody molecules, both specificand generalized affinity techniques are applicable. The most specificchoice of ligand for the affinity purification of an antibody is theantigen (or an epitope thereof) to which desired antibody reacts. Manyof the well-known immunosorbent assays such as the enzyme-linkedimmunosorbent assays (ELISA) are predicated on such specificantigen/antibody affinity interactions.

However, generalized affinity techniques are also useful. For example,Staphylococcal Protein A is known to bind certain antibodies of the IgGclass (See: Ey, P. L. et al. Immunochemistry 15:429-36 (1978)).Alternatively, antisera raised in heterologous species (e.g. rabbitanti-mouse antisera) can be used to separate general groups ofantibodies. (See, Current Protocols in Molecular Biology Supra, Chap11.)

Hydrophobic interaction chromatography was first developed following theobservation that proteins could be retained on affinity gels whichcomprised hydrocarbon spacer arms but lacked the affinity ligand.Although in this field the term hydrophobic chromatography is sometimesused, the term hydrophobic interaction chromatography (HIC) is preferredbecause it is the interaction between the solute and the gel that ishydrophobic not the chromatographic procedure. Hydrophobic interactionsare strongest at high ionic strength, therefore, this form of separationis conveniently performed following salt precipitations or ion exchangeprocedures. Elution from HIC supports can be effected by alterations insolvent, pH, ionic strength, or by the addition of chaotropic agents ororganic modifiers, such as ethylene or propylene glycol. A descriptionof the general principles of hydrophobic interaction chromatography canbe found in U.S. Pat. No. 3,917,527 and in U.S. Pat. No. 4,000,098. Theapplication of HIC to the purification of specific proteins isexemplified by reference to the following disclosures: human growthhormone (U.S. Pat. No. 4,332,717), toxin conjugates (U.S. Pat. No.4,771,128), antihemolytic factor (U.S. Pat. No. 4,743,680), tumornecrosis factor (U.S. Pat. No. 4,894,439), interleukin-2(U.S. Pat. No.4,908,434), human lymphotoxin (U.S. Pat. No. 4,920,196) and lysozymespecies (Fausnaugh, J. L. and F. E. Regnier, J. Chromatog. 359:131-146(1986)) and soluble complement receptors (U.S. Pat. No. 5,252,216). HICin the context of high performance liquid chromatography (HPLC) has beenused to separate antibody fragmens (e.g., F(ab′)₂) from intact antibodymolecules in a single step protocol. (Morimoto, K. et al., J. Biochem.Biophys. Meth. 24:107-117 (1992)).

In addition to affinity and HIC techniques, one or more of thetraditional protein purification schemes have been applied to antibodypurification. For example, Hakalahti, L. et al., (J. Immunol. Meth.117:131-136 (1989)) disclose a protocol employing two successive ionexchange chromatographic steps or one employing a single ion exchangestep followed by a HIC step. Danielsson A. et al. (J. Immunol. Methods115:79-88 (1988)) compare single step protocols based on anion exchange,cation exchange, chromatofocusing and HIC respectively.

Although Protein A affinity column chromatography is widely used, it isalso appreciated that elution of antibody from such columns can resultin leaching of residual Protein A from the support. Size exclusion HPLC(Das et al., Analytical Biochem, 145:27-36 (1985)) and anion exchangechromatography (EPO345549, published Dec. 13, 1989) have been suggestedas means for dealing with this problem.

It has now been surprisingly discovered that HIC can be usefullyemployed to remove contaminating Protein A from IgG mixtures eluted fromProtein A chromatographic support.

This invention relates to the application of HIC to the separation ofmonomeric IgG from mixtures containing same and to the integration ofHIC into a protocol combining Protein A and ion exchange chromatographyfor the purification of immunoglobulin G molecules.

BRIEF DESCRIPTION OF THE INVENTION

This invention relates to a method for separating IgG monomers fromaggregates in mixtures containing same by contacting said mixture with ahydrophobic interaction chromatographic support and selectively elutingthe monomer from the support.

In another aspect the invention provides for the purification of an IgGantibody from conditioned cell culture medium containing same comprisingsequentially subjecting the medium to (a) Protein A, (b) ion exchangechromatography, and (c) hydrophobic interaction chromatography.

In another aspect the invention provides a method for removing Protein Afrom a mixture comprising Protein A and antibodies comprising contactingsaid mixture with a hydrophobic interaction chromatography support andselectively eluting the antibody from the support.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a flow diagram of one process for purifying anantibody according to this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to protein purification techniques which haveapplication to the large scale purification of immunoglobulin molecules.The invention is particularly useful because it permits the recovery ofmonomeric IgG of >95% protein purity. The invention may be applied tothe purification of a number of different immunoglobulin G molecules.

Antibody-like proteins are proteins which may be purified by theprotocol described herein, such protocol being modified if necessary byroutine, non-inventive adjustments that do not entail undueexperimentation. Such proteins include isotypes. allotypes and allelesof immunoglobulin genes, truncated forms, altered antibodies, such aschimeric antibodies, humanized antibodies and the like, chemicallymodified forms such as by PEG treatment, and fusion proteins containingan immunoglobulin moiety. These proteins are referred to asantibody-like because they possess or retain sufficient immunoglobulinprotein properties (e.g. F_(c) determinants) to admit to purification bythe process of this invention. Unless specifically identified otherwise,the term antibody or immunoglobulin protein also includes antibody-likeproteins.

The immunoglobulin molecules of this invention can be isolated from anumber of sources, including without limitation, serum of immunizedanimals, ascites fluid, hybridoma or myeloma supernatants, conditionedmedia derived from culturing a recombinant cell line that expresses theimmunoglobulin molecule and from all cell extracts of immunoglobulinproducing cells. This invention is particularly useful for thepurification of antibodies from conditioned cell culture media of avariety of antibody producing recombinant cell lines. Although one mayexpect some variation from cell line to cell line and among the variousantibody products, based on the disclosure herein, it is well within thepurview of one of ordinary skill in this an to adapt the inventionherein to a particular combination of antibody protein and producingcell line.

Generally, genes encoding proteins such as antibodies may be cloned byincorporating DNA sequences coding for the desired regions of thepolypeptide into a recombinant DNA vehicle (e.g., vector) andtransforming or transfecting suitable prokaryotic or eukaryotic hosts.Suitable prokaryotic hosts include but are not limited to Escherichia,Streptomyces, Bacillus and the like. Suitable eukaryotic hosts includebut are not limited to yeast, such as Saccharomyces and animal cells inculture such as VERO, HeLa, mouse C127, Chinese hamster ovary (CHO),WI-38, BHK, COS, MDCK, myeloma, and insect cell lines. Particularlypreferred hosts are CHO cell lines deficient in dihydrofolate reductasesuch as ATCC CRL 1793, CRL 9096 and other cell lines described hereinbelow. Such recombinant techniques have now become well known and aredescribed in Methods in Enzymology, (Academic Press) Volumes 65 and 69(1979), 100 and 101 (1983), and the references cited therein. Anextensive technical discussion embodying most commonly used recombinantDNA methodologies can be found in Maniatis, et al., Molecular Cloning,Cold Spring Harbor Laboratory (1982) or Current Protocols in MolecularBiology, Greene Publishing, Wiley Interscience (1988,1991,1993).

One way of obtaining a DNA fragment encoding a desired polypeptide suchas an antibody molecule is via cDNA cloning. In this process, messengerRNA (mRNA) is isolated from cells known or suspected of producing thedesired protein. Through a series of enzymatic reactions, the mRNApopulation of the cells is copied into a complementary DNA (cDNA). Theresulting cDNA is then inserted into cloning vehicles and subsequentlyused to transform a suitable prokaryotic or eukaryotic host. Theresulting cDNA “library” is comprised of a population of transformedhost cells, each of which contain a single gene or gene fragment. Theentire library, in theory, provides a representation sample of thecoding information present in the mRNA mixture used as the startingmaterial. The libraries can be screened using nucleic acid or antibodyprobes in order to identify specific DNA sequences. Once isolated, theseDNA sequences can be modified or can be assembled into complete genes.

Specific fragments of an antibody gene can be engineered independentlyof the rest of the gene. DNA fragments encoding ComplementarityDetermining Regions (CDRs) can be integrated into DNA frameworksequences from heterologous species to yield altered antibodies. Thesealtered antibodies have significant utility in the treatment ofundesirable physiological conditions. For example, PCT/GB91/01554(published as WO92/04381) discloses the production of “humanized”antibodies useful for the treatment and prevention of RespiratorySyncytial Virus (RSV) infection. Alternatively, the entire variableregion of antibody gene can be fused to the constant domain of a secondantibody to form an altered antibody otherwise known as a “chimericantibody”. For example, PCT/US92/06194 (published as WO93/02108)discloses a monkey/human chimeric antibody reactive with the human CD4receptor.

Once the antibody gene or gene fragment has been cloned, the DNA may beintroduced into an expression vector and that construction used totransform an appropriate host cell. An expression vector ischaracterized as having expression control sequences as defined herein,such that when a DNA sequence of interest is operably linked thereto,the vector is capable of directing the production of the product encodedby the DNA sequence of interest in a host cell containing the vector.With specific reference to this invention, it is possible to assemblefragments of a single coding sequence such that upon expression anantibody molecule is formed. A particularly efficacious application ofthis protocol to recombinant antibody production is found in the Harris,et al. PCT Applications WO92/04381, published Mar. 19, 1992, citedabove, and in the Newman et al. PCT Application WO93/02108, publishedFeb. 4, 1993, cited above.

After the recombinant product is produced it is desirable to recover theproduct. If the product is exported by the cell producing it, theproduct can be recovered directly from the cell culture medium. If theproduct is retained intracellularly, the cells must be physicallydisrupted by mechanical, chemical or biological means in order to obtainthe intracellular product.

In the case of a protein product, the purification protocol should notonly provide a protein product that is essentially free of otherproteins, by which is meant at least 80% and preferably greater than 95%pure with respect to total protein in the preparation, but alsoeliminate or reduce to acceptable levels other host cell contaminants,DNA, RNA, potential pyrogens and the like. Furthermore, in the contextof antibody production by recombinant expression system, it isappreciated that aggregation of the 150,000 dalton IgG product intohigher molecular weight species can occur. Accordingly, for purposes ofproduct purity and standardization it is also useful to separate thenative 150,000 dalton monomeric species from higher molecular weightaggregates and other misfolded forms. While it is appreciated that the150,000 dalton IgG species is composed of four polypeptide chains (2heavy chains and 2 light chains), the 150,000 dalton species is referredto herein as a “monomer” or “monomeric IgG”.

As mentioned above, a variety of host cells may be used for theproduction of the antibodies of this invention. The choice of aparticular host cell is well within the purview of the ordinary skilledartisan taking into account, inter alia, the nature of the antibody, itsrate of synthesis, its rate of decay and the characteristics of therecombinant vector directing the expression of the antibody. The choiceof the host cell expression system dictates to a large extent the natureof the cell culture procedures to be employed. The selection of aparticular mode of production, be it batch or continuous, spinner or airlift, liquid or immobilized can be made once the expression system hasbeen selected. Accordingly, fluidized bed bioreactors, hollow fiberbioreactors, roller bottle cultures, or stirred tank bioreactors, withor without cell microcarriers may variously be employed. The criteriafor such selection are appreciated in the cell culture art. They are notdetailed herein because they are outside the scope of this invention.This invention relates to the purification of antibodies given theirexistence in a conditioned cell culture medium, hybridoma supernatant,antiserum, myeloma supernatant or ascites fluid.

As mentioned above this invention relates, inter alia, to application ofhydrophobic interaction chromatography (HIC) to the separation andpurification of antibody molecules. Hydrophobic molecules in an aqueoussolvent will self-associate. This association is due to hydrophobicinteractions. It is now appreciated that macromolecules such as proteinshave on their surface extensive hydrophobic patches in addition to theexpected hydrophilic groups. HIC is predicated, in part, on theinteraction of these patches with hydrophobic ligands attached tochromatographic supports. A hydrophobic ligand coupled to a matrix isvariously referred to herein as an HIC support, HIC gel or HIC column.It is further appreciated that the strength of the interaction betweenthe protein and the HIC support is not only a function of the proportionof non-polar to polar surfaces on the protein but by the distribution ofthe non-polar surfaces as well and the chemistry of the HIC support.

A number of chromatographic supports may be employed in the preparationof HIC columns, the most extensively used are agarose, silica andorganic polymer or co-polymer resins. Useful hydrophobic ligands includebut are not limited to alkyl groups having from about 2 to about 8carbon atoms, such as a butyl, propyl, or octyl; or aryl groups such asphenyl. Conventional HIC products for gels and columns may be obtainedcommercially from suppliers such as Pharmacia LKB AB, Uppsala, Swedenunder the product names butyl-SEPHAROSE®, phenyl or butyl-SEPHAROSE®CL-4B, butyl-SEPHAROSE® FF, octyl-SEPHAROSE® FF and phenyl-SEPHAROSE®FF; Tosoh Corporation, Tokyo, Japan under the product names TOYOPEARLether 650, phenyl 650 or butyl 650 (Fractogel); Miles-Yeda, Rehovot,Israel under the product name alkyl-agarose, wherein the alkyl groupcontains from 2-10 carbon atoms, and J. T. Baker, Phillipsburg, N.J.under the product name Bakerbond WP-HI-propyl.

It is also possible to prepare the desired HIC column using conventionalchemistry. (See: for example, Er-el. Z. et al. Biochem. Biophys. Res.Comm. 49:383 (1972) or Ulbrich, V. et al. Coll. Czech. Chem. Commun.9:1466 (1964)).

Ligand density is an important parameter in that it influences not onlythe strength of the interaction but the capacity of the column as well.The ligand density of the commercially available phenyl or octyl phenylgels is on the order of 40 μmoles/ml gel bed. Gel capacity is a functionof the particular protein in question as well as pH, temperature andsalt type and concentration but generally can be expected to fall in therange of 3-20 mg/ml of gel.

The choice of a particular gel can be determined by the skilled artisan.In general the strength of the interaction of the protein and the HICligand increases with the chain length of the alkyl ligands but ligandshaving from about 4 to about 8 carbon atoms are suitable for mostseparations. A phenyl group has about the same hydrophobicity as apentyl group, although the selectivity can be quite different owing tothe possibility of pi-pi orbital interaction with aromatic groups on theprotein. Selectively may also be affected by the chemistry of thesupporting resin.

Adsorption of the proteins to a HIC column is favored by high saltconcentrations, but the actual concentrations can vary over a wide rangedepending on the nature of the protein and the particular HIC ligandchosen. Various ions can be arranged in a so-called soluphobic seriesdepending on whether they promote hydrophobic interactions (salting-outeffects) or disrupt the structure of water (chaotropic effect) and leadto the weakening of the hydrophobic interactions. Cations are ranked interms of increasing salting out effect asBa++<Ca++<Mg++<Li+<Ca+<Na+<K+<R-b+<NH₄+, while anions may be ranked interms of increasing chaotropic effect asPO₄—<SO₄—<CH₃COO-<Cl⁻<Br-<NO₃-<ClO₄-<I-<SCN-. Accordingly, salts may beformulated that influence the strength of the interaction as given bythe following relationship:(NH₄)₂SO₄>Na₂SO₄>NaCl>NH₄Cl>NaBr->NaSCNIn general, salt concentrations of between about 0.75 and about 2Mammonium sulfate or between about 1 and 4M NaCl are useful.

The influence of temperature on HIC separations is not simple, althoughgenerally a decrease in temperature decreases the interaction. However,any benefit that would accrue by increasing the temperature must also beweighed against adverse effects such an increase may have on thestability of the protein.

Elution, whether stepwise or in the form of a gradient, can beaccomplished in a variety of ways: (a) by changing the saltconcentration, (b) by changing the polarity of the solvent or (c) byadding detergents. By decreasing salt concentration adsorbed proteinsare eluted in order of increasing hydrophobicity. Changes in polaritymay be affected by additions of solvents such as ethylene or propyleneglycol or (iso)propanol, thereby decreasing the strength of thehydrophobic interactions. Detergents function as displacers of proteinsand have been used primarily in connection with the purification ofmembrane proteins.

Although it has been discovered that HIC chromatography can be usedalone to separate monomeric IgG (MW 150,000) from aggregates andmisfolded species, as mentioned above, HIC is particularly useful whenused in combination with other protein purification techniques. That isto say, it is preferred to apply HIC to mixtures that have beenpartially purified by other protein purification procedures. By the term“partially purified” is meant a protein preparation in which the proteinof interest is present in at least 5 percent by weight, more preferablyat least 10% and most preferably at least 45%. By the term “mixture” ismeant the desired monomeric IgG antibody molecule in combination withundesirable contaminants such as, without limitation, one or more of:immunoglobulin aggregates, misfolded species, host cell protein, residuematerial from preceding chromatographic steps such as Protein A whenemployed. Accordingly, the application of HIC can also be appreciated inthe context of an overall purification protocol for immunoglobulinproteins such as affinity purified monoclonal antibodies. It has beenfound to be useful, for example, to subject a sample of conditioned cellculture medium to partial purification prior to the application of HIC.By the term “conditioned cell culture medium” is meant a cell culturemedium which has supported cell growth and/or cell maintenance andcontains secreted product. A sample of such medium is subjected to oneor more protein purification steps prior to the application of a HICstep. The sample may be subjected to affinity chromatography employingStaphylococcus Protein A as a first step. For example, PROSEP-A®(BioProcessing Ltd., U.K.) which consists of Protein A covalentlycoupled to controlled pore glass can be usefully employed. Other usefulProtein A formulations are Protein A SEPHAROSE® Fast Flow (Pharmacia)and TOYOPEARL 650M Protein A (TosoHaas). As a second step, ion exchangechromatography may be employed. In this regard various anionic orcationic substituents may be attached to matrices in order to formanionic or cationic supports for chromatography. Anionic exchangesubstituents include diethylaminoethyl(DEAE), quaternary aminoethyl(QAE)and quaternary amine(Q) groups. Cationic exchange substituents includecarboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) andsulfonate(S). Cellulosic ion exchange resins such as DE23, DE32, DE52,CM-23, CM-32 and CM-52 are available from Whatman Ltd. Maidstone, Kent,U.K. SEPHADEX®-based and cross-linked ion exchangers are also known. Forexample, DEAE-, QAE-, CM-, and SP-SEPHADEX® and DEAE-, Q-, CM-andS-SEPHAROSE® and SEPHAROSE® Fast Flow are all available from PharmaciaAB. Further, both DEAE and CM derivitized ethylene glycol-methacrylatecopolymer such as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or Mare available from Toso Haas Co., Philadelphia, Pa. Because elution fromion exchange supports usually involves addition of salt and because, asmentioned previously, HIC is enhanced under increased saltconcentrations, the introduction of a HIC step following an ionicexchange chromatographic step or other salt mediated purification stepis particularly preferred. Additional purification protocols may beadded including but not necessarily limited to further ionic exchangechromatography, size exclusion chromatography, viral inactivation,concentration and freeze drying.

For purposes of illustration only, this invention was applied to thepurification of several antibodies of the IgG isotype. Morespecifically, to a humanized antibody useful for the treatment of RSVinfection described by Harris et al.; 1992, Intl. Patent PublicationNumber WO/92/04381, published Mar. 19, 1992 (hereinafter “RSHZ-19”) anda chimeric antibody specifically reactive with the CD4 antigen describedby Newman et al. Int'l Patent Publication Number WO93/02108, publishedFeb. 4, 1993 (hereinafter CH-CD4). The construction of recombinantsystems for the production of RSHZ-19 and the CH-CD4 chimeric antibodiesare detailed in the above mentioned PCT Applications, the contents ofwhich are incorporated herein by reference for purpose of background andare summarized as follows.

An expression plasmid containing the RSHZ-19 coding sequence wascotransfected with pSV2dhfr into a dhfr-requiring Chinese Hamster Ovarycell line (CHO-DUXBII). The transfection was carried in growth mediumand employed the calcium coprecipitation/glycerol shock procedure asdescribed in: DNA Cloning, D. M. Glover ed. (Chap. 15, C. Gorman).Following transfection, the cells were maintained in growth medium for46 hours under growth conditions (as described above) prior to theselection procedure.

The selection and co-amplification procedure was carried out essentiallyas described by R. J. Kaufman, et al. (Mol. Cell. Biol. 5:1750-1759(1985)). Forty-six hours post transfection the cells were changed toselective medium MEM ALPHA (041-02571), 1% stock glutamine, 1% stockpen/strep (043-05070) and dialyzed bovine fetal calf serum (220-6300AJ)(Gibco, Paisley, Scotland). The cells were maintained in the selectivemedium for 8-10 days until dhfr+ colonies appeared. When the colonieswere established the cells were changed into a selective mediumcontaining methotrexate (A6770, Sigma Chem. Co., St. Louis, Mo.). Themethotrexate concentration was initially 0.02 μM and was increasedstepwise to 5 μM. During the amplification procedure aliquots of growthmedium from growing cells were assayed for RSHZ-19 production by humanIgG. Any antibody secreting recombinant cell line may be used to supplythe conditioned medium for purification according to this invention, aparticular cell line certainly is not required.

A transfected CHO cell line capable of producing RSHZ-19 can be culturedby a variety of cell culture techniques. For the application of thisinvention the particular method of culturing is not critical.

As mentioned previously, the particular recombinant production systemand the particular cell culturing protocol is outside the scope of thisinvention. The system and protocol discussed above are representative ofthe many options available to the skilled artisan and they are includedherein for purposes of illustration only. The purification protocolwhich is the subject of this invention is applicable, with only routinemodification, to a variety of recombinant antibodies and antibody-likeproteins regardless of how they are produced or cultured. For example achimeric monoclonal antibody to CD4 was also purified by the process ofthis invention.

The purified antibodies obtained by practicing the process of thisinvention have the following properties: 1) greater than 97% antibodyprotein by weight; 2) stable to proteolytic degradation at 4° C. for atleast three months; 3) low (<0.1 E.U./mg protein) endotoxin; 4) low (<1pg/mg protein) DNA; 5) non-antibody protein <5% by weight; and 6)virally inactive. The following examples further illustrate thisinvention but are not offered by way of limitation of the claims herein.

EXAMPLE 1 INTRODUCTION

The procedure outlined below was developed for the isolation andpurification of a monoclonal antibody against Respiratory SyncytialVirus (RSV). This antibody is a “humanized” IgG expressed in CHO cells,and grown in a stirred tank bioreactor. The antibody is more fullydescribed in PCT WO92/04381 and is otherwise referred to herein as RSHZ19. The process is designed to prepare RSHZ-19 of >95% purity whileremoving contaminants derived from the host cell, cell culture medium,or other raw materials. The process in its most preferred embodimentconsists of three purification steps (Protein A affinity, cationexchange, and hydrophobic interaction chromatography), two viralinactivation steps, and a diafiltration step to exchange the productinto a final buffer of choice (outlined in FIG. 1). All steps arecarried out at room temperature (18°-25° C.). All buffers are preparedwith WFI and filtered through either a 0.2 micron filter or a 10,000MWCO membrane before use. Buffer formulations are listed in Table 1.Tables 2, 4, 6 and 8 show the column parameters for examples IA, IB, ICand ID respectively. Tables 3, 5 and 7 and 9 provide a purificationsummary for examples IA, IB, IC and ID respectively.

The first step in the process (Protein A affinity chromatography onProSep A) can be rapidly cycled to accommodate varying amounts ofcell-free culture fluid (CCF), and has a capacity of approximately 15grams RSHZ-19 per liter of ProSep A. For example, 500 liters CCFcontaining 400-500 grams of IgG can be processed in 5 or 6 cycles. Thedownstream steps of the process (Cation Exchange Chromatography (CEC)and Hydrophobic Interaction Chromatography (HIC) are scaled toaccommodate approximately 130-140 grams RSHZ-19 per cycle. Thus, a 500liter culture containing 400-500 grams of RSHZ-19 is processed in threedownstream cycles after capture on ProSep A.

The hydrophobic interaction chromatography step (HIC) has beendemonstrated to remove residual Protein A that leaches from the ProteinA column during elution (See: examples IA-D). In addition, aggregates ofIgG can be removed over HIC, as shown in examples IC and ID.

The process description is normalized for any scale; linear flow rateslisted are independent of column diameter, loading ratios are in massper unit column volume. Examples are provided for the operation and therecovery at 1 gram, 40 gram, and 125 gram scales. (Examples IA-ID).

Purification Process Description

Removal of Cells from Culture

To harvest the culture fluid, the cells are removed using atangential-flow microfiltration device (Prostak) equipped with a 0.65micron filter or equivalent. The product is recovered in the permeate.For small volume cultures, centrifugation can be used.

Affinity Capture by Protein A Chromatography

The IgG is recovered from the CCF by adsorption chromatography on acolumn of ProSep A (BioProcessing Ltd.) previously equilibrated withPBS. The medium is applied to the column at a flow rate up to 1000 cm/hrat a load ratio of up to 15 grams IgG per liter column volume. Afterloading the column, it is washed with at least 3 column volumes of PBScontaining 0.1 M glycine. The RSHZ-19 is eluted with a low pH buffer byapplying approximately 3 column volumes of Elution Buffer.

The Protein A chromatography removes a large proportion of cell andmedia derived impurities (particularly protein and DNA in theflow-through and wash fractions), and concentrations RSHZ- 19 in theelution buffer for further processing.

Viral Inactivation at Acid pH (Optional)

The Protein A column eluate is collected and adjusted to pH 3.5 by theaddition of 2.5M HCl. The solution is transferred to a second vessel andheld at pH 3.5 for at least thirty minutes to provide viralinactivation, and readjusted to pH 5.5 by the addition of Tris buffer.The resulting solution is filtered through a prefilter (MilliporePolygard or equivalent) and a sterilized 0.2 μm filter (MilliporeMillipak or equivalent), and held in sterile containers at 4° C., orfrozen and held at −70° C.

The pH 3.5 treatment provides viral inactivation, and the pH 5.5adjustment prepares the solution for cation exchange chromatography(CEC). The pH 3.5 treatment can be omitted if desired.

Cation Exchange Chromatography

The pH inactivated Protein A eluate is further purified by CECchromatography on column of CM SEPHAROSE FF (Pharmacia LKB). The sampleis applied to the equilibrated column at a flow rate of 150 cm/hr and aload ratio of ≦20 grams protein per liter CM SEPHAROSE. After loading,the column is washed with 3 to 5 column volumes of Equilibration Buffer.The product is eluted with 3-5 column volumes of Elution Buffer.

The cation exchange chromatography step removes protein and non-proteinimpurities.

Viral Inactivation with Guanidine

The cation exchange eluate is adjusted to approximately 2.0M guanidinehydrochloride by the slow addition (with mixing) of one-half volume ofGuanidine Stock Solution. The rate of reagent addition is adjusted sothat it is added over a 5-15 minute period. The solution is transferredto a second vessel, and is held for thirty minutes to achieve viralinactivation. After holding, an equal volume of Ammonium Sulfate StockSolution is slowly added (with mixing), and the hydrophobic interactionchromatography (HIC) step is performed immediately. The rate of reagentaddition is adjusted so that it is added over a 5-15 minute period.

The guanidine treatment provides a second viral inactivation step, whenan acid inactivation step is employed, and keeps the RSHZ-19 solubleafter ammonium sulfate addition; the addition of ammonium sulfate servesto dilute the guanidine and prepare the solution for HIC.

Hydrophobic Interaction Chromatography

The guanidine-treated solution is further purified by application to anHIC column consisting of TOYOPEARL Phenyl-650M previously equilibratedwith Equilibration Buffer. The guanidine-treated solution is applied tothe column at a flow rate of 150 cm/hr and a load ratio of ≦20 gramsprotein per liter Phenyl-650M. After loading, the column is washed with3 to 5 column volumes of Equilibration Buffer. A linear gradient ofdecreasing ammonium sulfate is applied at a flow rate of 100-150 cm/hr,and the RSHZ-19 elutes as one major peak with impurities eluting laterin the gradient. The slope of the gradient is approximately 20 columnvolumes, starting at 100% Equilibration Buffer and ending at 100%Gradient Buffer (1.3 to 0M ammonium sulfate). The peak is collecteduntil the absorbance decreases to 20% of the maximum peak absorbance,then collection of the product fraction is ended. After the gradientends, the column is washed with approximately 3 column volumes of StripBuffer.

The HIC chromatography step removes additional protein and non-proteinimpurities, most notably residual Protein A, IgG aggregates, and hostDNA.

Concentration, Diafiltration and Final Filtration

The HIC elute is concentrated to approximately 10 milligram permilliliter using a tangential-flow ultrafiltration device (such as aMillipore CUF) outfitted with a 30,000 molecular weight cut-off filter,diafiltered into a suitable formulation buffer and filtered through asterilized 0.2 micron filter (Millipore Millipak or equivalent) intosterilized containers.

In-Process Assays

Process intermediates are assayed for total protein concentration byOD280 or Bradford assay, RSHZ-19 concentration by HLPC, sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Aggregatedproduct is assessed by size exclusion HPLC on TSK3000 SWXL, and ProteinA residue is assayed using an ELISA.

Pooling Criteria

The eluate fractions from the Protein A capture and cation exchangesteps are pooled based on the UV tracing on the chromatogram, and theentire peak is collected. The eluate from the HIC step is pooled basedon the UV tracing, and the main peak is pooled until the UV reading onthe tailing side of the peak reaches 20% of the peak maximum. The HICtail fraction contains the majority of the Protein A and aggregatedIgG's.

TABLE 1 Buffer Formulations Buffer Name Composition PBS 20 mM sodiumphosphate, 150 mM sodium chloride, pH 7 PBS/glycine PBS plus 0.1Mglycine ProSep Elution Buffer 25 mM citrate, pH 3.5 CM SEPHAROSE 10 mMcitrate, pH 5.5 Equilibration Buffer CM SEPHAROSE 40 mM citrate, 100 mMElution Buffer sodium chloride, pH 6 Guanidine Stock Solution 6Mguanidine hydrochloride, 50 mM sodium phosphate, pH 7 2.6M AmmoniumSulfate 2.6M ammonium sulfate, 50 mM Stock Solution sodium phosphate, pH7 2.0M Ammonium Sulfate 2.0M ammonium sulfate, 50 mM Stock Solutionsodium phosphate, pH 7 Phenyl-650 Equilibration 1.3M ammonium sulfate,50 mM Buffer sodium phosphate, pH 7 Phenyl-650 Gradient Buffer 50 mMsodium phosphate, pH 7 Butyl-650 Equilibration Buffer 1.0M ammoniumsulfate, 50 mM sodium phosphate, pH 7 Butyl-650 Gradient Buffer 50 mMsodium phosphate, pH 7 HIC Strip Buffer 0.2M NaOHExample IA. RSHZ-19 Purification at 1 Gram Scale Using TOYOPEARLPhenyl-650M

A 5.0 liter (20 cm diameter by 16 cm length) ProSep A affinity columnwas equilibrated with PBS (see Table 1) at 5.2 liter/min. 100 liters ofconditioned culture medium containing 0.8 grams per liter of RSHZ-19monoclonal antibody was clarified by microfiltration as described above,and applied to the column at a flow rate of 5.2 liter/min. After theload, approximately 15 liters of PBS/glycine was applied to the columnat the same flow rate. The IgG was eluted by applying 15-20 liters ofProSep A elution buffer. Fractions of the non-bound peak and the elutionpeak were collected and assayed for IgG content using an HPLC assay. Theeluate was approximately 15 liters in volume, and containedapproximately 5 milligrams protein per milliliter.

Immediately after elution, the sample was adjusted to pH 3.5 by theaddition of 2.5M hydrochloric acid, held for approximately 30 minutes,and adjusted to pH 5.5 by the addition of approximately 350 millilitersof 1M Tris base. After neutralizing to pH 5.5, the sample was filteredthrough a 0.1 micron Polygard CR filter in tandem with a sterile 0.2micron Millipak 200, into a sterile container. The filtrate was storedat 4° C. Samples of the filtrate were analyzed for IgG content using anHPLC assay, and for total protein by absorbance at 280 nanometers. Thesamples were also analyzed for Protein A content by an ELISA procedure.This pH 3.5 treated and filtered Prosep A eluate was used as the CMSEPHAROSE load in Examples IA, B and C.

400 milliliters of pH 3.5 treated and filtered ProSep A eluate wereloaded directly onto a 220 milliliter (4.4 cm diameter × 15 cm length)column of CM SEPHAROSE FF at 38 mL/min, which had been previouslyequilibrated with CM Equilibration buffer. After loading, the column waswashed at 38 mL/min with approximately 700 milliliters of CMEquilibration Buffer. The IgG was eluted by applying CM Elution Bufferat 38 mL/min. The IgG came of the column after approximately 1 bedvolume of Elution Buffer had passed. The entire peak was collected as CMSEPHAROSE eluate. Fractions of the CM non-bound, eluate and stripfractions were collected and analyzed for IgG content, total proteincontent, and Protein A content as described previously. The eluate wasapproximately 160 milliliters in volume, and contained approximately 12milligrams protein per milliter. This CM SEPHAROSE eluate was split intotwo equal portions of approximately 80 milliliters, and was used inExamples IA and IB for the HIC load.

To 80 milliliters of CM SEPHAROSE eluate was added (slowly with constantstirring) a total of 40 milliliters of Guanidine Stock Solution. Thisbrought the guanidine concentration to 2M for viral inactivation. Whilestirring the guanidine-treated solution, a total of 120 milliliters of2.6M Ammonium Sulfate Stock Solution was added. The resulting solutionwas 1.0M in guanidine and 1.3M in ammonium sulfate. The ammonium sulfatetreated solution was applied to an 80 mL column (3.2 cm diameter×10 cmlength) of TOYOPEARL Phenyl-650M, previously equilibrated with PhenylEquilibration Buffer. The flow rate was 20 mL/min throughout the run.After loading, the column was washed with approximately 350 millilitersof Phenyl Equilibration Buffer. The IgG was eluted by applying a lineargradient starting at 85% Equilibration/15% Gradient buffer and ending at0% Equilibration/100% Gradient buffer, in 18-19 column volumes. Thisrepresents a starting ammonium sulfate concentration of approximately1.1M and an ending concentration of 0M. The slope of this gradient wasapproximately a 4.7% increase in elution buffer per column volume, or−0.061M ammonium sulfate per column volume. The IgG began to elute fromthe column at approximately 7 column volumes and ended at approximately13 column volumes into the gradient (ammonium sulfate concentration ofapproximately 0.7 to 0.3M). The eluted fraction was collected until theUV absorbance on the tailing side of the peak decreased to 20% of thepeak height, then collection was switched to another vessel (tailfraction). At the end of the gradient, approximately 250 mL of HIC StripBuffer was applied to regenerate the column. Fractions of the Phenylnon-bound, eluate, tail and strip fractions were collected and analyzedfor IgG content, total protein content, and Protein A content asdescribed previously. The eluate was approximately 300 milliliters involume, and contained approximately 2.4 milligrams protein per milliter.

Table 2 summarizes the column parameters for this example. The productand protein recovery data for each step are shown in Table 3, along withthe Protein A content, expressed as nanograms Protein A per milligramIgG (ng/mg). As seen in Table 3, the Protein A reduction overPhenyl-650M is approximately 4-fold, and the recovery is approximately94%.

TABLE 2 Column Parameters at 1 gram scale using Phenyl-650M ColumnColumn Volume dia × length Load Flow Rates Step (liter) (cm) Ratio (cm/hr) (mL/min) ProSep A 5.0  20 × 16 16.0 g IgG per 1000 5200 liter bedvolume CM SEPHAROSE FF 0.22 4.4 × 15  9.1 g protein per 150 38 liter bedvolume Phenyl-650M 0.08 3.2 × 10 10.4 g protein per 150 20 liter bedvolume

TABLE 3 Purification Summary for Example IA, 1 gram scale usingPhenyl-650M Total RSHZ- Total Step Protein Volume 19^(a) Protein^(b)Yield A^(c) Step (Liters) (Grams) (Grams) (%) (ng/mg) Cell-free 100 80.3n.d. — 0 Culture Fluid ProSep A 15.8 73.8 80.4 92 20.2 Eluate CM SEPHA-(0.4)^(d) (1.87)^(d) (2.04)^(d) ROSE^(d) Load CM SEPHA- 0.16 20.1 1.88100  14.5 ROSE Eluate Phenyl-650M^(d) (0.21)^(d) (0.72)^(d) (0.83)^(d)Load Phenyl-650M 0.31 0.68 0.73 94 3.5 Eluate (Phenyl Tail 0.59 0.0750.10 — 133)^(e) Cumulative 86 Recovery (%) ^(a)by HPLC ^(b)by Absorbenceat 280 nm ÷ 1.27 mL mg⁻¹ cm⁻¹ ^(c)by ELISA ^(d)Only a portion of thetotal eluate from the previous column was carried forward, as describedin the text above ^(e)Protein A migrates primarily in the Tail fractionExample IB. RSHZ-19 Purification at 1 Gram Scale Using TOYOPEARLButyl-650M

This preparation used the same CM SEPHAROSE eluate as described inExample IA, and the HIC step was performed using TOYOPEARL Butyl-650Minstead of Phenyl-650M. The preparation of the CM SEPHAROSE eluate isdescribed in Example IA above. To 80 milliliters of CM SEPHAROSE eluatewas added (slowly with constant stirring) a total of 40 milliliters ofGuanidine Stock Solution. This brought the guanidine concentration to 2Mfor viral inactivation. While stirring the guanidine-treated solution, atotal of 120 milliliters of 2.0M Ammonium Sulfate Stock Solution wasadded. The resulting solution was 1.0M in guanidine and 1.0M in ammoniumsulfate. The ammonium sulfate treated solution was applied to a an 80 mLcolumn (3.2 cm diameter×10 cm length) of TOYOPEARL Butyl-650M,previously equilibrated with Butyl Equilibration Buffer. The flow ratewas 20 mL/min throughout the run. After loading, the column was washedwith approximately 350 milliliters of Butyl Equilibration Buffer. TheIgG was eluted by applying a linear gradient starting at 65%Equilibration/35% Gradient buffer and ending at 20% Equilibration/80%Gradient buffer, in 12-13 column volumes. This represents a startingammonium sulfate concentration of approximately 0.65M and an endingconcentration of approximately 0.2M. The slope of this gradient wasapproximately a 3.3% increase in elution buffer per column volume, or−0.033M ammonium sulfate per column volume. The IgG began to elute fromthe column at approximately 2 column volumes and ended at approximately9 column volumes into the gradient (ammonium sulfate concentration ofapproximately 0.58 to 0.35M). The eluate fraction was collected untilthe UV absorbance on the tailing side of the peak decreased to 10% ofthe peak height, then collection was switched to another vessel. At theend of the gradient, approximately 250 mL of Butyl Gradient Buffer wasapplied, and a small peak eluted and was collected. Approximately 250 mLof HIC Strip Buffer was applied to regenerate the column. Fractions ofthe Butyl non-bound, eluate, tail and strip fractions were collected andanalyzed for IgG content, total protein content, and Protein A contentas described previously. The eluate was approximately 400 milliliters involume, and contained approximately 1.5 milligrams protein per milliter.

Table 4 summarizes the column parameters for this example. The productand protein recovery data for each step are shown in Table 5, along withthe Protein A content, expressed as nanograms Protein A per milligramIgG (ng/mg). Although the recovery of IgG is lower compared to ExampleIA (79% v. 94%), the Protein A content is reduced approximately 20-foldusing Butyl-650M as an HIC step.

TABLE 4 Column Parameters at 1 gram scale using Butyl-650M Column ColumnVolume dia × length Load Flow Rates Step (liter) (cm) Ratio (cm/hr)(mL/min) CM SEPHAROSE FF 0.22 4.4 × 15  9.1 g protein per 150 38 literbed volume Butyl-650M 0.08 3.2 × 10 10.4 g protein per 150 20 liter bedvolume

TABLE 5 Purification Summary for Example IB, 1 gram scale usingButyl-650M Total RSHZ- Total Step Protein Volume 19^(a) Protein^(b)Yield A^(c) Step (Liters) (Grams) (Grams) (%) (ng/mg) Cell-free 100 80.3n.d. — 0 Culture Fluid ProSep A 15.8 73.8 80.4 92 20.2 Eluate CM SEPHA-(0.4)^(d) (1.87)^(d) (2.04)^(d) ROSE^(d) Load CM SEPHA- 0.16 2.01 1.88100  14.5 ROSE Eluate Butyl-650M^(d) (0.21)^(d) (0.76)^(d) (0.86)^(d)Load Butyl-650M 0.41 0.60 0.62 79 0.7 Eluate (Butyl Tail 0.40 0.03 0.03— 31.4)^(e,f) (Butyl Strip 0.53 0.10 0.10 — n.d.)^(g) Cumulative 73Recovery Mass Balance 95 (% of load) ^(a)by HPLC ^(b)by Absorbence at280 nm ÷ 1.27 mL mg⁻¹ cm⁻¹ ^(c)by ELISA ^(d)Only a portion of the totaleluate from the previous column was carried forward, as described above^(e)Protein A migrates primarily in the Tail fraction ^(f)Tail contains3% of the load ^(g)strip contains 13% of the protein that was loadedExample IC. RSHZ-19 Purification at 40 Gram Scale Using TOYOPEARLPhenyl-650M

This preparation used the same ProSep A eluate as described in ExampleIA, and the downstream steps were scaled-up to accommodate approximately40 grams of protein, using TOYOPEARL Phenyl-650M as the HIC medium. Thepreparation of the CM SEPHAROSE Load is described in Example IA above.7.8 liters of pH 3.5 treated and filtered ProSep A eluate were loadeddirectly onto a 4.2 liter (25 cm diameter×8.5 cm length) column of CMSEPHAROSE FF which had been previously equilibrated with CMEquilibration buffer at 1.2 L/min. After loading, the column was washedat 1.2 L/min with approximately 8 liters of CM Equilibration Buffer. TheIgG was eluted by applying CM Elution Buffer at 1.2 L/min. The IgG cameoff the column after approximately 1 bed volume of Elution Buffer hadpassed. The entire peak was collected as CM SEPHAROSE eluate. The columnfractions of the CM non-bound and eluate were collected and analyzed forIgG content, total protein content, and Protein A content as describedpreviously. The eluate was approximately 5.7 liters in volume, andcontained approximately 6-7 milligrams protein per milliter.

To 5.6 liters of CM SEPHAROSE eluate was added (slowly with constantstirring) a total of 2.8 liters of Guanidine Stock Solution (3.2kilogram by weight). This brought the guanidine concentration to 2M forviral inactivation, at a volume of 8.3 liters. While stirring theguanidine-treated solution, a total of 8.3 liters (9.7 kg by weight) of2.6M Ammonium Sulfate Stock Solution was added. The resulting solutionwas 1.0M in guanidine and 1.3M in ammonium sulfate, with a final volumeof 16.7 liters. The ammonium sulfate treated solution was applied to a4.6 liter column (18 cm diameter×18 cm length) of TOYOPEARL Phenyl-650M,previously equilibrated with Phenyl Equilibration Buffer. The flow ratewas 0.5-0.6 L/min throughout the run. After loading, the column waswashed with approximately 14 liters of Phenyl Equilibration Buffer. TheIgG was eluted by applying a linear gradient starting at 100%Equilibration Buffer and ending at 100% Gradient buffer, in 20 columnvolumes. This represents a starting ammonium sulfate concentration ofapproximately 1.3M and an ending concentration of 0M. The slope of thisgradient was approximately a 5% increase in elution buffer per columnvolume, or −0.065M ammonium sulfate per column volume. The IgG beganeluting from the column at approximately 7 column volumes and ended atapproximately 12 column volumes into the gradient (ammonium sulfateconcentration of approximately 0.85 to 0.5M). The eluate fraction wascollected until the UV absorbance on the tailing side of the peakdecreased to 20% of the peak height, then collection was switched toanother vessel (tail). Fractions of the Phenyl non-bound, eluate andtail and strip fractions were collected and analyzed for IgG content,total protein content, and Protein A content as described previously.The eluate was approximately 15.4 L in volume, and containedapproximately 2.2 milligrams protein per milliter.

The Phenyl Eluate was concentrated to approximately 16 mg/mL using atangential flow ultrafiltration apparatus (CUF, Millipore Corp.)equipped with 30,000 MWCO Omega membranes (Filtron Corp.) and bufferexchanged by continuous diafiltration against a suitable formulationbuffer.

Table 6 summarizes the column parameters for this example. The productand protein recovery data for each step are shown in Table 7, along withthe Protein A content, expressed as nanograms Protein A per milligramIgG (ng/mg) and the IgG aggregate content, expressed as % of total IgG.As seen in Table 7, the Protein A reduction over Phenyl-650M isapproximately 3-fold, and the recovery is approximately 90%. IgGaggregates were reduced from 0.5% in the CM SEPHAROSE eluate to 0.06% inthe formulated product.

TABLE 6 Column Parameters at 40 gram scale Column Column Volume dia ×length Load Flow Rates Step (liter) (cm) Ratio (cm/hr) (L/min) CMSEPHAROSE FF 4.2  25 × 8.5 8.9 g protein per 150 1.2 liter bed volumePhenyl-650M 4.6 18 × 18 10.4 g protein per 140 0.6 liter bed volume

TABLE 7 Purification Summary for Example IC: 40 gram scale Total TotalStep IgG Volume RSHZ-19^(a) Protein^(b) Yield Protein A^(c) AggregateStep (Liters) (Grams) (Grams) (%) (ng/mg) (%) Cell-free Culture 100 80.3n.d. — 0 n.d. Fluid ProSep A 15.8 73.8 80.4 92 20.2 n.d. Eluate CMSEPHAROSE^(d) (7.84)^(d) (36.0)^(d) (37.3)^(d) Load CM SEPHAROSE 5.7136.5 37.3 100  21.4 0.5% Eluate Phenyl-650M 15.4 32.8 33.6 90 8.0 <0.05Eluate (Product) Phenyl Tail 24.6 2.5 3.0 — 78.0 5.1)^(e) Formulated 2.032.8 32.0 100  6.5 0.06 Product Cumulative Recovery 83 (%) Mass Balance(% of load) 97 ^(a)by HPLC ^(b)by Absorbence at 280 nm ÷ 1.27 mL mg⁻¹cm⁻¹ ^(c)by ELISA ^(d)Only a portion of the total ProSep A Eluate wascarried forward ^(e)Protein A and IgG aggregates migrate primarily inthe tail fractionExample ID. RSHZ-19 Purification at 125 Gram Scale Using TOYOPEARLPhenyl-650M

A 5.5 liter (20 cm diameter by 18 cm length) ProSep A affinity columnwas equilibrated with PBS (see Table 1) at 4.8 liter/min. 450 liters ofconditioned culture medium containing 0.94 grams per liter of RSHZ-19monoclonal antibody was clarified by microfiltration as described above,and applied in four separate 90-95 liter portions and one 40 literportion to the column at a flow rate of 4.8 liter/min (and sothroughout). Each cycle on the column ran as follows: After the load,approximately 17 liters of PBS/glycine was applied to the column at thesame flow rate. The IgG was eluted by applying 15-20 liters of ProSep AElution buffer. Fractions of the non-bound peak and the elution peakwere collected and assayed for IgG content using an HPLC assay. Theeluate from each cycle was approximately 9 liters in volume, andcontained approximately 5-10 milligrams protein per milliliter.Immediately after elution, the ProSep A eluates were adjusted to pH 3.5by the addition of 2.5M hydrochloric acid, held for approximately 30minutes, and adjusted to pH 5.5 by the addition of approximately 250milliliters of 1M Tris base. After neutralizing to pH 5.5, the eluateswere pooled together, and filtered through a 0.1 micron Polygard CRfilter in tandem with a sterile 0.2 micron Millipak 200, in 5 literaliquots in sterile containers. The filtrate was stored at 4° C. Samplesof the filtrate were analyzed for IgG content using an HPLC assay, andfor total protein by absorbance at 280 nanometers. The samples were alsoanalyzed for Protein A content by an ELISA procedure, and IgG aggregatesby HPLC.

The downstream steps were scaled-up to accommodate approximately 120-140grams of protein. 16.3 liters of pH 3.5 treated and filtered ProSep Aeluate containing approximately 130 grams of protein was loaded directlyonto a 14.4 liter (35 cm diameter×15 cm length) column of CM SEPHAROSEFF at 2.4 L/min, which had been previously equilibrated with CMEquilibration buffer. After loading, the column was washed at 2.4 L/minwith approximately 45 liters of CM Equilibration Buffer. The IgG waseluted by applying CM Elution Buffer at 2.4 L/min. The IgG began toelute from the column after approximately 1-2 bed volumes of ElutionBuffer had passed. The entire peak was collected as CM SEPHAROSE eluate.Fractions of the CM non-bound and eluate were collected and analyzed forIgG content, total protein content, and IgG aggregate. The eluate wasapproximately 21 liters in volume, and contained approximately 120 gramsprotein.

To 19.4 liters of CM SEPHAROSE eluate was added (slowly with constantstirring) a total of 9.7 liters of Guanidine Stock Solution. Thisbrought the guanidine concentration to 2M for viral inactivation, at avolume of 29.1 liters. While stirring the guanidine-treated solution, atotal of 29.1 liters of 2.6M Ammonium Sulfate Stock Solution was added.The resulting solution was 1.0M in guanidine and 1.3M in ammoniumsulfate, with a final volume of 58.2 liters. The ammonium sulfatetreated solution was applied to a 12.4 liter column (30 cm diameter×18cm length) of TOYOPEARL Phenyl-650M, previously equilibrated with PhenylEquilibration Buffer. The flow rate was 1.1-1.3 L/min throughout therun. After loading, the column was washed with approximately 37 litersof Phenyl Equilibration Buffer. The IgG was eluted by applying a lineargradient starting at 80% Equilibration Buffer/20% Gradient Buffer andending at 20% Equilibration Buffer/80% Gradient buffer, in 12 columnvolumes. This represents a starting ammonium sulfate concentration ofapproximately 1.0M and an ending concentration of approximately 0.26M.The slope of this gradient was approximately a 5% increase in Gradientbuffer per column volume, or −0.065M ammonium sulfate per column volume.The IgG came off the column essentially in the middle of the gradient,with the peak containing approximately 0.8M ammonium sulfate. The eluatefraction was collected until the UV absorbance on the tailing side ofthe peak decreased to 20% of the peak height, then collection wasswitched to another vessel (tail). Fractions of the Phenyl non-bound,eluate and tail and strip fractions were collected and analyzed for IgGcontent, total protein content, and IgG aggregate. The eluate wasapproximately 29 L in volume, and contained approximately 100 gramsprotein.

The Phenyl Eluate was concentrated to approximately 10 mg/mL using atangential flow ultrafiltration apparatus (CUF, Millipore Corp.)equipped with 30,000 MWCO Omega membranes (Filtron Corp.) and bufferexchanged by continuous diafiltration against a suitable formulationbuffer. The product was analyzed for IgG content, total protein, ProteinA and IgG aggregate.

Table 8 summarizes the column parameters for this example. The productand protein recovery data for each step are shown in Table 9, along withthe Protein A content, expressed as nanograms Protein A per milligramIgG (ng/mg) and the IgG aggregate content, expressed as % of total IgG.As seen in Table 9, the Protein A reduction over CM SEPHAROSE andPhenyl-650M is approximately 7-fold, and the cumulative recovery isapproximately 70%. IgG aggregates were reduced from 0.4% in the CMSEPHAROSE eluate to 0.06% in the formulated product.

TABLE 8 Column Parameters at 125 gram scale Column Column Volume dia ×length Load Flow Rates Step (liter) (cm) Ratio (cm/hr) (L/min) ProSep A5.0 20 × 18 14-16 g RSHZ-19 915 4.8 per liter bed volume CM SEPHAROSE FF14.4 35 × 15 8.4 g protein per 150 2.4 liter bed volume Phenyl-650M 12.430 × 18 8.6 g protein per 100 1.2 liter bed volume

TABLE 9 Purification Summary for Example ID: 125 gram scale Total TotalStep IgG Volume RSHZ-19^(a) Protein^(b) Yield Protein A^(c) AggregateStep (Liters) (Grams) (Grams) (%) (ng/mg) (%) Cell-free Culture 416 392 477^(C) — 0 n.d. Fluid ProSep A 48.7 375 384 96 11.7 0.4 Eluate CMSEPHAROSE^(d) (16.3)^(d)  (125)^(d)  (129)^(d) — Load CM SEPHAROSE 20.6116 117 93 n.d. 0.4 Eluate Phenyl-650M 29.1   98.1   99.8 85 n.d. <0.05Eluate Formulated 9.29   89.8   91.1 92 1.7 0.06 Product CumulativeRecovery 70 (%) ^(a)by Reversed-Phase HPLC ^(b)by Absorbence at 280 nm ÷1.27 mL mg⁻¹ cm⁻¹ ^(c)by Bradford assay ^(d)Downstream process capacityis approximately 140 grams; only a portion of the total ProSep A eluateis carried forward

TABLE 10 Purity analysis: 125 Gram Scale Aggregates Purity^(a)Activity^(b) Step (% of Total IgG) (% of Total Area) (%) CCF notapplicable not done   80^(c) ProSep Eluate 0.4 98.5 105 CM Eluate 0.498.4 109 Phenyl Eluate <0.05 98.3  99 Final product 0.06 99.7 115^(a)Determined by scanning densitometry of reducing SDS-PAGE; sum ofarea of Heavy and Light chains of IgG ^(b)Calculated ratio of activity(determined by Bovine RS Virus binding ELISA) to RSHZ-19 concentration(determined by A280) ^(c)Calculated ratio of activity, by bovine RSvirus ELISA, to RSHZ-19 concentration by HPLC

EXAMPLE II

The anti-CD4 monoclonal antibody CH-CD14 was made by cell culturetechniques and partially purified using Protein A and ion exchangechromatography in a fashion similar to that used in Example I, exceptthat an anion exchange resin was employed. An Amicon column (1 cmdiameter by 10 cm high) was packed with 8 mL of Phenyl TOYOPEARL 650Mresin (lot #65PHM01 H). The flow rate was maintained at 2 mL/min for allsteps. The column was equilibrated with 20 mL Equilibration buffer (1Mammonium sulfate, 50 mM sodium phosphate, pH 7.0). The product wasprepared for loading on the column by taking 5 mL partially purifiedCH-CD4 monoclonal antibody (17 mg/mL concentration by absorbance at 280nm), adding 2.5 mL 6M guanidine HCl, 50 mM sodium phosphate, pH 7.0,mixing, holding for 30 minutes, and then adding 7.5 mL 2M ammoniumsulfate, 50 mM sodium phosphate, pH 7.0. The final ammonium sulfateconcentration of the load was 1M, the final guanidine HCl concentrationwas 1M, the final volume after samples were taken was 12 mL, and thefinal concentration of product was 5.6 mg/mL. This material was thenloaded on the column at 2 mL/min and then eluted with a 10 column volumelinear gradient of Equilibration buffer to 50 mM sodium phosphate, pH7.0. Fractions of approximately 4 mL were taken as the product elutedfrom the column.

The results are shown in Table 11. All of the product eventually elutedin the gradient. Protein A also eluted and was enriched in laterfractions from the gradient. To achieve a reduction in Protein A in thefinal product, it was necessary to exclude some of the fractions at theend of the gradient, thus reducing the yield of product. A two-foldreduction in Protein A was possible with an 86% yield of product and athree-fold reduction was possible with a 50% yield.

TABLE 11 Cumulative Specific Cumulative Protein A Fraction VolumeProduct Protein A Protein A Product Removal No. (mL) (mg/mL) (ng/mL)(ng/mg) Yield Factor^(a) 0 Load 5.8 200.7 35 12 Eluate 1 5.5 0.04 0.0 0 0% 2 3 0.31 0.0 0  2% 3 4.2 1.32 6.4 4 10% 8.6 4 4.2 3.1 16.9 5 28% 7.05 4 4 68.3 10 51% 3.3 6 4.2 3.4 80.9 14 72% 2.4 7 4.2 2.3 94.1 19 86%1.9 8 4.2 1.3 79.3 22 94% 1.6 9 4.2 0.71 49.9 24 98% 1.4 10 4.2 0.3632.8 26 100%  1.3 11 4 0.19 22.9 27 101%  1.3 12 4 0.11 13.5 27 102% 1.3 13 9.6 0.05 8.5 28 102%  1.2 ^(a)Removal factor is calculated bypooling eluate fractions from start of eluate to desired fraction anddividing initial Protein A in load by Protein A ng/mg in pooled eluatefractions. For example, the factor 8.6 is calculated by dividing the sumof Protein A in fractions 1-3 by the sum of product in fractions 1-3 toget ng/mg. This number is then divided by 35 ng/mg in the load to obtain8.6.

EXAMPLE IIB:

CH-CD4 monoclonal antibody was partially purified, and prepared forloading on the HIC column as described in example IIA. The same column,flowrate, equilibration and loading described in example IIA were used.In this example, after the column was loaded and washed withEquilibration buffer, it was washed with 18 mL Wash buffer (1M ammoniumsulfate, 50 mM sodium citrate, pH 3.5). This was followed by anotherwashing with 13.5 mL Equilibration buffer (described in example IIA).The column was eluted with a gradient and then washed with water asdescribed in example IIA. The column eluate was divided into 14 mLfractions which were then analyzed for product and Protein A.

The results are presented in Table 12. Protein A could be reduced by 6fold at 90% yield, and by 8 fold at 78% yield.

TABLE 12 Cumulative Specific Cumulative Protein A Fraction VolumeProduct Protein A Protein A Product Removal No. (mL) (mg/mL) (ng/mL)(ng/mg) Yield Factor¹ 0 Load 5.80 167.1 29 12 Eluate 1 14 1.50 0 0.0 30%— 2 14 2.35 13.6 3.5 78% 8.1 3 14 0.38 8.3 5.2 85% 5.6 4 14 0.18 0 5.089% 5.8 5  9 0.13 0 4.9 91% 5.9 ¹As in footnote “a” to Table 11.

Comparing the results of example IIB with example IIA it can be seenthat Protein A was reduced by 6 to 8 fold with about 80% yield when thepH 3.5 wash was included, but reduction was only about 2 fold with 80%yield of CH-CDH without the pH 3.5 wash.

EXAMPLE IIC:

This example was performed similar to example IIB, except that the scalewas increased. Equilibration and Wash buffers are described in exampleIIB. The column was 5 cm in diameter and 28 cm high and the flowrate was50 mL/min. CH-CD4 monoclonal antibody was prepared and partiallypurified, as described in example IIA. The partially purified product(440 mL) was mixed with 220 mL 6M guanidine HCl for 31 min. Then, 660 mL2M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 was added. The finalammonium sulfate concentration was 1M and the final antibodyconcentration was 5.3 mg/mL. After sampling, the load volume was 1,290mL.

The column was equilibrated with 2 column volumes of Equilibrationbuffer and then loaded on the column. The column was then washed with630 mL of Equilibration buffer, 1,000 mL of Wash buffer, 800 mL ofEquilibration buffer, and then eluted with a 5 column volume gradientfrom 0.75M ammonium sulfate, 50 mM sodium phosphate, pH 7.0, to 50 mMsodium phosphate, pH 7.0. Fractions were collected during elution.

The results are presented in Table 13. Protein A was reduced 100 foldwith a yield of 70%, or by 30 fold with an antibody yield of 80%.

TABLE 13 Cumulative Specific Cumulative Protein A Fraction VolumeProduct Protein A Protein A Product Removal No. (mL) (mg/mL) (ng/mL)(ng/mg) Yield Factor^(a) 0 Load 5.30 198 37 1290  Eluate 1 581 0.03 00.0  0% 2 304 3.60 0 0.0 16% 3 243 5.40 0 0.0 35% 4 238 4.50 0 0.0 51% 5237 3.40 2.4 0.1 63% 282 6 239 2.2 4.7 0.4 71% 107 7 256 1.2 4.8 0.6 75%66 8 252 0.7 7.6 0.9 78% 41 9 240 0.4 5.3 1.1 79% 33 10 250 0.4 5.7 1.481% 27 11 202 0.4 4.38 1.5 82% 25 12 210 1 148.5 6.8 85% 5

EXAMPLE IID

Partially purified CH-CD4 monoclonal antibody was prepared as shown inexample IIA. Equilibration and Wash buffers are described in exampleIIB. Four milliliters of 6M guanidine HCl, 50 mM sodium phosphate, pH7.0 was added to 8 mL of a 9.5 mg/mL solution of partially purifiedCH-CD4 monoclonal antibody and incubated for 30 minutes. Then, 12 mL of2M ammonium sulfate, 50 mM sodium phosphate, pH 7.0 was added slowly.The column was 0.5 cm in diameter and 20 cm high (4 mL) and the flowratefor all steps was 0.5 mL/min. The column was rinsed with 2 columnvolumes each of water and Equilibration buffer. Then, 22 mL of the loadsolution was passed through the column, followed by 2 column volumes ofEquilibration buffer, followed by Wash buffer until the pH of the columneffluent was 3.5. This was followed by Equilibration buffer until theeffluent pH was 7.0. The column was eluted with 0.3M ammonium sulfate,50 mM sodium phosphate, pH 7.0. After the UV trace started to rise, 12.6mL of eluate were collected and analyzed for product and Protein A. Theyield of product was 80% and the Protein A was reduced from 28 ng/mg to6 ng/mg, a reduction of 4.7 fold.

1. A method for purifying monomeric IgG antibody from a mixture comprising said monomeric antibody and at least one of immunoglobulin aggregates, misfolded species, host cell protein or protein A comprising contacting said mixture with a hydrophobic interaction chromatographic support and selectively eluting the monomer from the support.
 2. The method according to claim 1 A method for purifying monomeric IgG antibody from a mixture comprising said monomeric IgG antibody and at least one of immunoglobulin aggregates, misfolded species, and protein A, wherein said method comprises the steps of: (i) contacting said mixture with a hydrophobic interaction chromatographic support and (ii) selectively eluting the monomeric IgG antibody from the support wherein the monomeric IgG is selected from the group consisting of anti-RSHZ-19 and CH-CD4.
 3. The method according to claim 1 wherein the HIC support is selected from the group consisting of alkyl_(C2-C8) agarose, aryl-agarose, alkyl-silica, aryl-silica alkyl organic polymer resin and aryl organic polymer resin.
 4. The method according to claim 3 wherein the support is selected from the group consisting of butyl-, phenyl-, and octyl-agarose and butyl-, phenyl- and ether- organic polymer resin.
 5. The method according to claim 4 wherein the support is phenyl-organic polymer resin.
 6. The method according to claim 4 wherein the support is butyl-organic polymer resin.
 7. The method according to claim 1 wherein the antibody is selectively eluted with a low salt buffer.
 8. The method according to claim 7 2 wherein the antibody is selectively eluted with a gradient decreasing in salt to 50 mM phosphate, pH7.0.
 9. A method for the purification of an IgG antibody from conditioned cell culture medium containing same comprising sequentially subjecting the medium to (a) Protein A affinity chromatography, (b) ion exchange chromatography, and (c) hydrophobic interaction chromatography.
 10. The method according to claim 9 wherein the ion exchange chromatography employs a support selected from the group consisting of CM-23—, CM-32-, CM-52- cellulose; CM-and, SP-cross-linked dextrans, CM- and S-argose; CM-organic polymer resin; DEAE-QAE-Q-cross-linked dextians; DEAE-,QAE-,Q- linked agarose; and DEAE organic polymer resins and is by a buffered salt solution.
 11. The method according to claim 10 wherein the support is CM-agarose Fast Flow and the salt is NaCl.
 12. The method according to claim 10 wherein the buffered salt solution is 40 mM citrate containing, 100 mM, NaCl, pH 6.0.
 13. The method according to claim 9 wherein the hydrophobic interaction chromatographic employs a support selected from the group consisting of alkyl_(C2-C8)-agarose, aryl-agarose, alkyl-silica, aryl-silica, alkyl-organic polymer resin and aryl-organic polymer resin.
 14. The method according to claim 13 wherein the support is selected from the group consisting of butyl-, phenyl- and octyl-agarose and butyl-, phenyl- and ether-organic polymer resin.
 15. The method according to claim 14 wherein the support is phenyl-organic polymer resin or butyl-organic polymer resin.
 16. The method according to claim 9 wherein the support is phenyl- or butyl-organic polymer resin and the antibody is selectively eluted with a low salt buffer.
 17. The method according to claim 16 wherein the antibody is selectively eluted with a gradient decreasing to 50 mM sodium phosphate buffer, pH 7.0.
 18. The method according to claim 9 wherein the Protein A chromatography employs as a support Protein A linked to controlled pore glass and elution is by a low pH buffer.
 19. The method according to claim 18 wherein said buffer is 25 mM citrate, pH 3.5.
 20. A method for purifying antibody from a conditioned cell medium comprising: (a) adsorbing the antibody onto a Protein A chromatographic support; (b) washing the adsorbed antibody with at least one buffer; (c) eluting the antibody from step (b); (d) adsorbing the antibody from step (c) onto an ion exchange chromatographic support; (e) washing the absorbed antibody with at least one buffer; (f) selectively eluting the antibody from step (e); (g) adsorbing the eluate of step (f) onto a hydrophobic interaction chromatographic support; (h) washing the adsorbed antibody with at least one buffer; (i) eluting the adsorbed antibody; and (j) recovering the antibody.
 21. The method according to claim 20 which includes one or more optional steps of inactivating viruses if present.
 22. The method according to claim 21 wherein a viral inactivation step is performed after step (f) and before step (g).
 23. The method according to claim 22 wherein said viral inactivation step comprises treatment of the eluate with guanidine hydrochloride for a period of time sufficient to inactivate virus followed by the addition of an ammonium sulfate solution.
 24. The method according to claim 23 wherein the guanidine hydrochloride is present at 2.0M and following treatment eluate from step (f) is adjusted to 1.3M ammonium sulfate.
 25. The method according to claim 22 wherein an additional viral inactivation step is performed after step (c) and before step (d).
 26. The method according to claim 25 wherein said additional viral inactivation step comprises treatment of the eluate of step (c) with acid.
 27. The method according to claim 26 wherein the pH of the eluate is adjusted to pH 3.5 and maintained at that pH for a period of time sufficient to inactivate virus, and terminating the treatment by adjusting the pH to 5.5.
 28. The method according to claim 20 wherein the ion exchange support of step (d) is selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate(P), diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary (Q), substituted cellulosic resins, cross linked dextrans, agarose and organic polymer resins.
 29. The method according to claim 28 wherein the cationic support is CM-agarose.
 30. The method according to claim 20 wherein the hydrophobic interaction chromatographic support is selected from the group consisting of alkyl _(C2-C8)-agarose, aryl-agarose, alkyl-silica, aryl-silica, alkyl-organic polymer resin and aryl-organic polymer resins.
 31. The method according to claim 30 wherein the support is selected from the group consisting of butyl-, phenyl- and octyl-agarose and phenyl-, ether- and butyl-organic polymer resins.
 32. The method according to claim 31 wherein the support is phenyl- or butyl-organic polymer resins.
 33. The method according to claim 20 wherein said protein is recovered by pooling and concentrating the protein containing fractions from chromatography step (i) by ultrafiltration.
 34. The method according to claim 20 wherein the chromatographic support of step (a) is Protein A linked to controlled pore glass.
 35. The method according to claim 20 wherein the absorbed antibody of step (h) is washed with two buffers, a first equibration buffer and a second low pH wash buffer.
 36. The method according to claim 35 wherein the pH of the second buffer is less than 4.0.
 37. The method according to claim 36 wherein the second buffer is 1M ammonium sulfate, 50 mM sodium citrate, pH 3.5.
 38. A method of removing Protein A from a mixture comprising Protein A and IgG antibodies comprising contacting the mixture with a hydrophobic interaction chromatography support and selecting eluting the antibody from the support.
 39. The method according to claim 38 A method for removing Protein A from a mixture comprising Protein A and IgG antibodies comprising contacting said mixture with a hydrophobic interaction chromatography support and selectively eluting the antibody from the support which includes washing the support prior to elution with a buffer having a pH less than 7.0.
 40. The method according to claim 39 wherein the pH of the wash buffer is less than 4.0.
 41. The method according to claim 40 wherein the buffer is ( 1M ammonium sulfate, 50 mM sodium citrate, pH 3.5) . 